There is currently great interest in the potential use of carbon nanoscale materials for medical and biological applications. This interest is at least partially fueled by the spherical or cylindrical surface morphology of many of these materials, particularly those having cage-like nanostructures that are stable with respect to cage opening under in vivo environmental conditions. However, in order to become biocompatible such carbon nanomaterials need to be surface-functionalized with organic groups that can facilitate both improved solubility in physiological solutions and selective binding affinity to bio-targets. Therefore, developing simple and cost-effective chemical methods for covalent functionalization of carbon nanocage materials has become an area of immense fundamental and industrial importance. This research holds great promise for bio-medical applications—as was recently demonstrated by the ability of modified carbon nanotubes to cross the cell membrane and enter the nuclei of cells, and their being non-toxic to the cell at concentrations up to 10 μM [Pantarotto et al., J. Am. Chem. Soc. 2003, 125, 6160; Pantarotto et al., Chem. Commun. 2004, 16-17]. Using appropriate chemistry, a variety of biologically-active molecules can be covalently attached to carbon nanostructures.
Carbon nanotubes (CNTs, aka fullerene pipes) are nanoscale carbon structures comprising graphene sheets conceptually rolled up on themselves and closed at their ends by fullerene caps. Single-walled carbon nanotubes (SWNTs) comprise but a single such graphene cylinder, while multi-walled nanotubes are made of two or more concentric graphene layers nested one within another in a manner analogous to that of a Russian nesting doll. SWNT diameters generally range from 0.4 to 4 nm. These nanotubes can be from 100 nm to several micrometers (microns) long, or longer. Since their initial preparation in 1993 [lijima et al., Nature, 1993, 363, 603; Bethune et al., Nature, 1993, 363, 605; Endo et al., Phys. Chem. Solids, 1993, 54, 1841], SWNTs have been studied extensively due to their unique mechanical, optical, electronic, and other properties. For example, the remarkable tensile strength of SWNTs has resulted in their use in reinforced fibers and polymer nanocomposites [Zhu et al., Nano Lett. 2003, 3, 1107 and references therein].
SWNTs normally self-assemble into aggregates or bundles in which up to several hundred tubes are held together by van der Waals forces. For many applications, including bio-medical ones, the separation of individual nanotubes from these bundles is essential. Such separation improves the dispersion and solubilization of the nanotubes in the common organic solvents and/or water needed for their processing and manipulation. Covalent modifications of the SWNT surface generally help to solve this problem by improving the solubility and processability of the nanotubes. While chemical functionalizations of the nanotube ends generally do not change the electronic and bulk properties of these materials, sidewall functionalizations do significantly alter the intrinsic properties of the nanotubes. However, the extent of documented results in this new field of chemistry is limited, primarily due to the current high cost of the nanotubes. Additional challenges faced in the modifications of SWNT sidewalls are related to their relatively poor reactivity—largely due to a much lower curvature of the nanotube walls relative to the more reactive fullerenes [M. S. Dresselhaus, G. Dresselhaus, P. C. Eklund, Science of Fullerenes and Carbon Nanotubes, Academic Press, San Diego, 1996, Vol. 1], and to the growing strain within the tubular structure with increasing number and size of functional groups attached to graphene walls. The sp2-bonding states of all the carbon atoms comprising the nanotube framework facilitate the predominant occurance of addition-type reactions. The best characterized examples of these reactions include additions to the SWNTs of nitrenes, azomethine ylides and aryl radicals generated from diazonium salts [V. N. Khabashesku, J. L. Margrave, Chemistry of Carbon Nanotubes in Encyclopedia of Nanoscience and Nanotechnology, Ed H. S. Nalwa, American Scientific Publishers, 2004; Bahr et al., J. Mater. Chem., 2002, 12, 1952; Holzinger et al., Angew Chem. Int. Ed., 2001, 40, 4002].
The first sidewall functionalization of SWNTs was accomplished by attaching fluorine groups, through direct fluorination, the result being fluoronanotubes [Mickelson et al., Chem. Phys. Lett., 1998, 296, 188]. These fluorinated nanotube derivatives were found to be soluble in alcohols and other polar solvents [Mickelson et al., J. Phys. Chem. B, 1999, 103, 4318]. Microscopy studies show the unroping of such fluoronanotubes to yield bundles with diameters ten times smaller than that seen for pristine SWNTs—thus resulting in their improved dispersion and processability.
It has been shown that the C—F bond in fluoronanotubes is rather weak and therefore fluorine can be substituted or removed using wet chemistry methods [Boul et al., Chem. Phys. Lett., 1999, 310, 367]. Applicants have used such an approach based on fluorine displacement reactions in fluoronanotubes to develop a group of methods for the production of amino- and hydroxyl-group terminated SWNT derivatives [V. N. Khabashesku, J. L. Margrave, Chemistry of Carbon Nanotubes in Encyclopedia of Nanoscience and Nanotechnology, Ed. H. S. Nalwa, American Scientific Publishers, 2004; Khabashesku et al., Acc. Chem. Res. 2002, 35 (12), 1087; Stevens et al., Nano Lett. 2003, 3, 331; Zhang et al., Chem. Mater. 2004, 16(11), 2055; and commonly assigned co-pending U.S. patent application Ser. No. 10/714,187, filed Nov. 14, 2003.
“Amino-nanotubes” have been prepared by heating fluoronanotube dispersions in diamines NH2(CH2)nNH2 (n=2−6) at 100° C. for 1-3 hrs in the presence of pyridine (Py) as a catalyst [Stevens et al., Nano Lett. 2003, 3, 331]. The presence of primary terminal amino groups in the prepared amino-nanotubes was established by a color reaction with ninhydrin (Kaiser test), used routinely in biochemistry on aminoacids and peptides, and by formation of C(═O)NH peptide linkages in the reaction with adipoyl chloride to produce a nylon-nanotube polymer material. Based on thermal gravimetric analysis (TGA) weight loss and energy-dispersive analysis of X-rays (EDAX) data, the degree of sidewall functionalization in such amino-nanotubes was estimated as being 1 functional group per 8 to 12 carbon atoms of the carbon nanotube.
Fluoronanotubes have also been used as precursors for the preparation of a series of “hydroxyl-nanotubes” by two simple and inexpensive methods. In the first method, fluoronanotubes are reacted with diols and triols pre-treated with LiOH. In the second method, the reactions with amino alcohols in the presence of pyridine are utilized [Zhang et al., Chem. Mater. 2004, 16(11), 2055]. The degree of sidewall functionalization in such “hydroxyl-nanotube” derivatives was estimated to be in the range of 1 functional group per every 15 to 25 nanotube carbons, depending upon the derivatization method and alcohol reagent used. The “hydroxyl-nanotubes” form stable suspensions/solutions in polar solvents, such as water, ethanol and dimethylformamide, which facilitate their improved processing in copolymers and in ceramics nanofabrication, and their compatibility with biomaterials.
Another novel approach to derivatizing (functionalizing) SWNTs involves the addition of functional organic radicals generated from acyl peroxides, e.g., succinic or glutaric acid peroxides to SWNT sidewalls. The “carboxyl-nanotubes” prepared by this method were characterized by subsequent reactions with SOCl2 and diamines to form amides, which presented the chemical evidence for covalent attachment of —COOH group-terminated carboxy-alkyl radicals to the SWNTs. Compared to pristine SWNTs, the “carboxyl-nanotubes” show an improved solubility in polar solvents, e.g., alcohols (1.25 mg/ml in iso-propanol) and water. The degree of SWNT sidewall functionalization with the carboxyl-terminated groups was estimated to be about 1 functional group per every 24 nanotube carbons, based on thermogravimetric-mass spectrometric (TG-MS) data [Peng et al., J. Am. Chem. Soc. 2003, 125, 15174; commonly assigned co-pending U.S. patent application Ser. No. 10/714,014, filed Nov. 14, 2003]. The attachment of functional groups to nanotube sidewalls has been directly verified by transmission electron microscopy (TEM) with images depicting “bumpy” and “hairy” surfaces of the single nanotubes.
The preparation of SWNTs derivatized with functional groups on their sidewalls permits their use in applications using their hydrogen bonding ability and the chemical reactivity of their respective terminal —NH2, —COOH and —OH groups in biomaterials, such as biosensors, vehicles for drug delivery, nanotube-reinforced biopolymers, and ceramics for tissue engineering and implants in orthopedics and dentistry. The sidewall functional groups, as well as the activated unsaturated carbon-carbon bonds on the nanotube surface, can also act as a free radical scavengers and can likely demonstrate a high antioxidant activity in aging treatment applications. The related experimental data on these nanosystems are yet unknown, while studies of the similarly functionalized fullerene C60 derivatives as antioxidants are already in progress. Methods of using such chemistry to directly incorporate carbon nanotubes into biological molecules, such as amino acids, could further extend such bio-medical applications of CNTs.